Particulate cartilage system

ABSTRACT

The present invention is directed to compositions having at least one neocartilage particle, juvenile cartilage particle or a combination thereof and a matrix, and methods and devices that include the compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/010,779, filed Dec. 13, 2004, which claimspriority from provisional patent application Ser. No. 60/528,865, filedDec. 11, 2003, the entire disclosure of which is herein incorporated byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

REFERENCE TO A SEQUENCE LISTING

Not Applicable.

BACKGROUND OF THE INVENTION

Injuries and damage to articular cartilage result in lesions in thecartilage that often lead to disability, pain and reduced or disturbedfunctionality. Historically there has been limited success in the repairof these injuries and lesions, (i.e. characterized by a repair thatre-establishes a structurally and functionally competent articularcartilage tissue of a lasting nature). Many injuries and defects toarticular cartilage penetrate the bone and bone-marrow spaces as well(i.e., an osteochandral defect).

Articular cartilage tissue has a tough and elastic character; it coversthe ends of bones in joints and enables the bones to move smoothly overone another. Numerous diseases, including osteoarthritis, and traumaticinjuries from activities and accidents cause damage to articularcartilage.

Articular cartilage lacks a direct blood supply, is aneural, alymphatic,and contains a single cell type, the chondrocyte. Its lack ofvascularization, high matrix-to-cell ratio and lack of a local source ofundifferentiated cell reserves results in a limited capacity toregenerate following injury or degenerative loss. Repair of damaged ordiseased mature articular cartilage historically has been difficultbecause of its very limited ability to self-repair. Adult humanarticular cartilage usually does not self-repair or only partially healsunder normal biological conditions.

In the past, repair interventions based on the use of adult human tissueor isolated chondrocyte autografts or allografts have not providedcompletely satisfactory results, from the standpoint of a restoration ofthe architecture of the articulating surface.

Grafting of pure articular cartilage alone has shown little or nosuccess, nor has the implantation of isolated cartilage flakes aftertraumatic dissociation or ablation without a bony support, as cartilagedoes not adhere to bony surfaces nor is bone able to facilitatecartilage fixation.

In vitro culture of chondrocytes under controlled conditions can giverise to normal articular cartilage tissue growth. Adkisson, U.S. Pat.Nos. 6,235,316 and 6,645,764. However, normal adult chondrocytesgenerally have lost their potential to reproduce and generate newcartilage in vivo, although they are responsible for maintaining tissuehomeostasis. Accordingly, there exists a need for improved compositionsand methods for repairing articular cartilage.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention is directed to compositionsincluding a cartilage or a neocartilage construct of juvenile cartilageparticles and biocompatible chondro-conductive/inductive matrix. Someembodiments may further include an osteo-conductive matrix. Thecartilage may be distributed throughout substantially all of thebiocompatible chondro-conductive matrix or just a portion of the matrix,the portion may range from 90 to 10%. In some embodiments thesurface-to-volume ratio of the cartilage particles is greater than 1. Inany embodiment the biocompatible chondro-conductive/inductive matrix maybe fibrinogen, fibrinogen/thrombin, albumin, in-situ formingpoly(ethylene glycol) (PEG) hydrogel, fibrin/hyaluronate,fibrin/collagen/hyaluronate, PEG/hyaluronate, PEG/collagen, other plasmaand protein-based adhesives and sealants, other natural adhesives andsealants and any combination thereof. In any embodiment the compositionmay further comprise an osteo-conductive matrix. The osteo-conductivematrix may be fibrinogen, fibrinogen/thrombin, fibrin/tri-calciumphosphate, fibrin/collagen/tri-calcium phosphate,fibrin/hyaluronate/tri-calcium phosphate, in-situ forming PEG hydrogelsealants, PEG/tri-calcium phosphate, PEG/collagen, demineralized bonematrix, and any combination thereof. In any embodiment the compositionmay include an associated matrix containing∥collagen|, polylactic acid(PLA) and polyglycolic acid (PGA).

In any embodiment the composition may include other cartilage tissues,such as costal cartilage, nasal cartilage, trachea cartilage, sternumcartilage and any other cartilage tissue that contains Collagen II andnot Collagen I and III.

Another aspect of the invention may include a composition containingneocartilage or juvenile cartilage particles from a non-autologoussource.

Another aspect of the invention is directed toward or includes methodsof using the inventive compositions for inducing articular cartilage(i.e., a chondral defect) formation, repairing articular cartilage orrepairing articular cartilage together with filling a bone defect invertebrates (i.e., an osteochondral defect). The methods includedisposing the inventive compositions in a site where regeneration,augmentation, the induction of articular cartilage formation, therepairing of articular cartilage or the repairing of articular cartilageand also filling a bone defect, is desired.

Another aspect of the invention includes a device including any of thecompositions of the invention and the device may also be used in amethod of articular cartilage repair by disposing the device in a defectin need of repair.

Yet another aspect of the invention includes a method of preparing anyof the compositions of the invention, scoring a surface of juvenilecartilage or neocartilage; separating at least a portion of the scoredcartilage from underlying bone; and adding a preservative to theseparated cartilage.

Another aspect of the invention includes a kit for repairing cartilageincluding any of the compositions of the invention, a pouch having ahollow interior; a sterile container positioned in the hollow interiorhaving a receptacle therein; and one or more particles of juvenilecartilage and/or neocartilage positioned in the receptacle of thecontainer.

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims and accompanying figures where:

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS AND FIGURES

FIG. 1 shows an embodiment of the invention wherein cartilage particlesare distributed throughout substantially all of the biocompatiblechondro-conductive/inductive matrix.

FIG. 2 shows an embodiment of the invention wherein cartilage particlesare distributed throughout approximately 75% or less of thebiocompatible chondro-conductive/inductive matrix.

FIG. 3 shows an embodiment of the invention wherein cartilage particlesare distributed throughout substantially all of the biocompatiblechondro-conductive/inductive matrix and further comprises a particulateosteo-conductive matrix.

FIG. 4 shows juvenile cartilage particles encapsulated within ahyaluronate hydrogel.

FIG. 5 shows the morphologic appearance of human juvenile cartilageparticles, pre-cast in a fibrin matrix, after 60 days of laboratoryculture.

FIG. 6 shows a repaired medial femoral condyle (right side ofphotograph) of a Spanish goat, 6 weeks after implantation of humanjuvenile cartilage particles with a fibrin matrix and live periostealflap.

FIG. 7 shows a 1 mm thick section through the defect site represented inFIG. 6

FIG. 8 shows viable human juvenile cartilage implanted into a goatfemoral condyle 6 weeks after surgery.

FIG. 9 shows the morphologic appearance of human juvenile cartilageparticles implanted into a goat femoral condyle 6 weeks after surgery.

DETAILED DESCRIPTION OF THE INVENTION

The term “juvenile cartilage” refers to a chondrocyte cell, cells,cartilage tissue, or progeny or derivatives thereof, that are committedto become cartilage, or progenitor cells which are capable of undergoingproliferation growth, differentiation and maturation into chondrocytesand formation of cartilaginous tissue. In general, such chondrocytes aremost readily found in tissue from individuals who encompass allograft,autograft and xenograft sources. In humans, preferably chondrocytes arefrom those less than fifteen years of age, and more preferably, lessthan two years of age. Typically, immature or juvenile chondrocytesexpress an enhanced ability to synthesize and organize a hyalinecartilage extra-cellular matrix. This activity usually is highest incells freshly isolated from donor tissue and decays during subsequentmanipulation such as passage and expansion.

The term “neocartilage” refers to cartilage characterized by one or moreof the following attributes: containing membrane phospholipids enrichedin Mead acid, containing membrane phospholipids depleted in linoleic orarachidonic acid, being substantially free of endothelial, bone and/orsynovial cells, having a sulfated glycosaminoglycan S-GAG content of atleast 400 mg/mg, positive for type II collagen expression, beingsubstantially free of type I, III and X collagen, containing a matrixsubstantially free of biglycan, having multiple layers of cells randomlyarranged, rather than separated into distinct zones of chondrocytematuration, being enriched in high molecular weight aggrecan, beingproduced in vitro and essentially free of non-cartilage material, orbeing characterized by having multiple layers of cells surrounded by asubstantially continuous insoluble glycosaminoglycan andcollagen-enriched hyaline extracellular matrix.

The term “biocompatible” refers to materials which, when incorporatedinto the invention, have acceptable toxicity, acceptable foreign bodyreactions in the living body, and acceptable affinity with livingtissues.

The term “chondro-inductive” refers to the ability of a material toinduce the proliferation, growth differentiation and/or other maturationof chondrocytes or chondroprogenitor cells and/or proliferation, growthdifferentiation and/or maturation of chondrocytes or chondroprogenitorcells or production of articular cartilage from neocartilage progenitorcells, chondrocytes or cartilage. A chondro-inductive material may actdirectly as a growth factor which interacts with precursor cells toinduce chondrocyte proliferation, growth differentiation and/ormaturation, or the material may act indirectly by inducing theproduction of other chondro-inductive factors, such as growth factors.This induction may optionally include without limitation signaling,modulating, and transforming molecules.

The term “chondro-conductive” refers to materials which provide anenvironment for proliferation, differentiation, growth, ingrowth and/ororientation of cartilage tissue, chondrocyte cells or chondroprogenitorcells from surrounding tissues.

The term “chondro-inductive/conductive” refers to the characteristic ofbeing both chondro-inductive and chondro-conductive.

The term “matrix” refers to substance(s) which adhered to or partiallyembedded within which something is contained.

The term “osteo-conductive” refers to materials which provide anenvironment for proliferation, differentiation, growth, ingrowth and/ororientation of osteogenic cells.

The term “flap” refers to an autologous or allogenic membrane of livecells, natural or synthetic material that can be vital or devitalized.The flap contains the matrix with cartilage particles that can beattached to natural cartilage or underlying bone in vivo by sutures orsutureless attachment such as chemical tissue welding or gluing, or byphysical attachment devices such as tacks or staples.

The compositions and methods as described herein comprise useful repairof damaged or diseased articular cartilage. The compositions and methodsinclude a cartilage matrix or particles and a biocompatiblechondro-conductive/inductive matrix.

In another aspect of the invention a device as described herein may bedisposed into a site of cartilage repair, regeneration or augmentation.

In another aspect of the invention, the compositions further comprise aparticulate osteo-conductive matrix.

In other embodiments the cartilage matrix comprises a cartilagegrowth-enhancing material selected from the group consisting of at leastone juvenile cartilage particle, at least one neocartilage particle, acombination thereof, and any of the above together with an associatedmatrix.

The compositions may be used according to the methods of the invention,for implanting or transplanting or otherwise disposing a reparativeconstruct into a site in need of articular cartilage repair,regeneration or growth.

In another aspect of the invention a device may be formed from theinventive compositions and the device may be disposed in a site in needof articular cartilage repair.

In some embodiments the compositions further comprise a particulateosteo-conductive matrix.

The biocompatible chondro-conductive/inductive matrix of the inventioncomprises any appropriate compound or combination of compounds that isinductive or conductive for the formation or repair of articularcartilage in the inventive compositions and methods.

The chondro-conductive/inductive matrix may comprise fibrinogen. Thefibrinogen may be from any suitable source. For example, one skilled inthe art will recognize that fibrinogen may be derived from blood bankproducts—either heterologous (pooled or single-donor) or autologouscryoprecipitate or fresh frozen plasma. Fibrinogen can also be derivedfrom autologous fresh or platelet-rich plasma, obtained using cell-saveror other techniques. U.S. Pat. No. 5,834,420 also discloses a method forobtaining fibrinogen.

In other embodiments the biocompatible chondro-conductive/inductivematrix comprises thrombin. The thrombin may be from any suitable source.One skilled in the art will recognize that thrombin can be isolated bywell known means or purchased commercially. See U.S. Pat. No. 4,965,203,and Berliner, J L, Thrombin: Structure and Function (Ed) Plenum PubCorp; (1992) for exemplary methods of isolation and/or purification.

In any embodiment the biocompatible chondro-conductive/inductive matrixmay comprise a combination of fibrinogen and thrombin. Thechondro-conductive/inductive matrix may contain equal proportions offibrinogen and thrombin or more of either fibrinogen than thrombin ormore thrombin than fibrinogen. When used in combination the two may bein any proportion, ranging from one part of either compared to theamount of the other up to equal proportions of each of the two.

Regardless of whether the fibrinogen or the thrombin are mixed with theneocartilage, juvenile cartilage or are separate components of thebiocompatible chondro-conductive/inductive matrix, when practicingcertain embodiments of the invention the fibrinogen and thrombincomponents preferably are kept separate from each other prior to thetime of use. The fibrinogen and the thrombin are then brought intocontact with each other at the time of use. A common type of applicatorthat may be used for this purpose consists of a double syringe, joinedby a Y-connector where the components mix as they emerge. This type ofapplicator, used with a blunt cannula, is useful for combining thethrombin and the fibrinogen and also useful in the methods of theinvention for disposing or transplanting the inventive compositions to asite wherein articular cartilage repair is desired. In cases where thearticular cartilage repair site is open for repair, the fibrinogenand/or thrombin can also be used with a spray attachment to coversurfaces; or the fibrinogen and/or thrombin may be applied to anabsorbable carrier or dressing, such as a cellulose sponge, collagenfleece, vital or devitalized periosteum or any other suitable means.

In various embodiments the chondro-conductive/inductive matrix maycomprise one or more of fibrinogen, thrombin, fibrinogen/thrombin(Tisseel or Crosseal), albumin, in-situ forming poly(ethylene glycol)(PEG) hydrogel, fibrin, hyaluronate, fibrin/hyaluronate, collagenhyaluronate, fibrin/collagen/hyaluronate, PEG/hyaluronate, PEG/collagen,PEG base sealants (CoSeal), or other plasma and protein-based adhesivesand/or sealants, other natural adhesives and/or sealants andcombinations thereof, that are biocompatible with regard to thearticular cartilage repair or replacement and are inductive orconductive for the cartilage matrix or cartilage growth-enhancingmaterial in the repair or replacement of articular cartilage.

The biocompatible chondro-conductive/inductive matrix, may in someembodiments optionally function to facilitate anchoring and/or fixationof the composition in the methods of the invention to repair the desiredarticular cartilage.

The invented compositions may also include materials which are not yetknown, but which provide characteristics relating to these componentswhich are similar to the materials described herein.

The cartilage tissue in certain embodiment of the inventive compositionalso may comprise neocartilage or juvenile cartilage or a combination ofneocartilage or juvenile cartilage. The neocartilage and juvenilecartilage may be in any proportion to each other, ranging from one cellor part of either compared to the other up to equal proportions of eachof the two. For example, the cartilage matrix or cartilagegrowth-enhancing material may contain equal proportions of neocartilageand juvenile cartilage or more of either neocartilage than juvenilecartilage or more juvenile cartilage than neocartilage. In someembodiments the compositions of the invention further comprise aparticulate osteo-conductive matrix. The neocartilage or juvenilecartilage is in the form of particles in the cartilage matrix orcartilage growth-enhancing material. The particles increase the surfaceto volume ratio in the cartilage matrix or cartilage growth-enhancingmaterial, which allows for improved integration and metabolite andgrowth factor exchange, which advantageously results in enhancedviability and shelf life for the compositions. The neocartilage andjuvenile cartilage particles may vary in size ranges |from| 1 to 27 mm³.Thus, the neocartilage and juvenile cartilage particles placed incartilage matrix or cartilage growth-enhancing material also may vary insize from single cells with associated matrix to 100 mm³ in sizedepending on application or defect type. For a somewhat typical defectof 2 cm, at least 1×10⁶ to 2×10⁶ cells would be disposed, preferably2×10⁶ to 4×10⁶, and most preferably 10×10⁶ to 20×10⁶. The amount ofcells used may vary depending on the specific circumstances of a defectin need of repair and the goals of the patient. For example, one skilledin the art would recognize that on average, adult tissue has about a 5to 10% cell mass per gram of tissue. This equates to about a 7% fill.However, some cell death will likely occur during maturation so a higherinitial cell count is typically preferable.

In terms of providing economic ratios of tissue to percentage fill ofdefects, to maximize tissue use, approximately 300 mg of tissue wouldprovide for about a 50% defect fill, although less, approximately 200mg, for a 30% defect fill, and most preferably, for a 10% defect fill,60 mg would be utilized.

The matrix portion of the cartilage matrix or cartilage growth-enhancingmaterial may comprise thrombin, fibrinogen, media or fibrinogen incombination with media or thrombin in combination with media. Anysuitable media may be used for the media component. Examples of suitablemedia include, but are not limited to a conditioned growth mediumadapted for use in growing cartilage cell cultures which containsheparin-binding growth factors, at least one of which is acartilage-derived morphogenetic protein (Chang et al., J. Biol Chem 269:28227-28234), other pre-conditioned medias, Dulbecco's modified Eagle'smedium (DMEM), Minimum Essential Medium and RPMI (Roswell Park MemorialInstitute) medium. The culture medium may also comprise ascorbate,and/or exogenous autocrine growth factors.

The juvenile cartilage in the invention may be from any suitable source.The juvenile cartilage or chondrocytes used in the composition may beharvested from donor tissue and prepared by dividing or mincing thedonor cartilage into small pieces or particles. The juvenile cartilageparticles may comprise juvenile cells or tissue, which may be intact,minced or disrupted, such as by homogenizing the tissue. Examples ofsources of donor cartilage include autologous, allogenic or xenogenicsources. In the case of autologous grafts, cartilage is harvested fromcartilaginous tissue of the patient's own body. Typical sources forautologous donor cartilage include the articular joint surfaces,intercostals cartilage, and cartilage from the ear or nasal septum. Inthe case of allografts, the cartilage may be taken from any appropriatenon-identical donor, for example from a cadaveric source, otherindividuals or a transgenic source or similar appropriate source.

In any embodiment of the invention the cartilage matrix or cartilagegrowth-enhancing material may comprise juvenile cartilage (withoutneocartilage) in any suitable tissue culture media. The juvenilecartilage may also comprise juvenile cartilage tissue in a matrix ofthrombin or juvenile cartilage in a matrix of fibrinogen.

In any embodiment that includes neocartilage, the cartilage matrix orcartilage growth-enhancing material may comprise neocartilage cells inany suitable tissue culture media. The neocartilage matrix or cartilagegrowth-enhancing material may also comprise neocartilage in a thrombinmatrix or neocartilage in a fibrinogen matrix.

In embodiments having neocartilage, the neocartilage may be from anysuitable source. The neocartilage particles may comprise neocartilagecells or tissue, which may be intact, minced or disrupted, such as byhomogenizing the tissue. The neocartilage may be either autologous orallogenic. Examples of suitable sources include commercially availablesources, such as Carticel® (Genzyme Biosurgery, Cambridge, Mass.),embryonic sources, tissue culture sources or any other suitable source.For example a cell culture may be produced to grow neocartilage byisolating immature chondrocytes, e.g., fetal, neonatal, andpre-adolescent chondrocytes from donor articular cartilage. Theneocartilage of the inventive cartilage matrix or cartilagegrowth-enhancing material may be obtained by culturing chondrocytesunder suitable culture conditions known in the art, such as growing thecell culture at 37 degrees C. in a humidified atmosphere with theaddition of 2-10% carbon dioxide, preferably 5%. Chondrocytes may beisolated by methods known in the art such as by sequential enzymedigestion techniques. The isolated chondrocytes may then be seededdirectly on a tissue culture vessel in any suitable media. Also see, forexamples of other sources, U.S. Pat. No. 5,326,357 which describesmethods to produce a continuous cartilaginous tissue and U.S. Pat. No.6,235,316 which discloses neocartilage compositions and uses, which areincorporated by reference, herein in their entirety.

The juvenile or neo cartilage tissue for the cartilage matrix orcartilage growth-enhancing material can be mammalian or avianreplacement tissue, most preferably from the same species as therecipient, for example human donor tissue for human replacement andequine tissue for equine use. Furthermore, mammalian replacement tissuecan be produced using chondrocytes from transgenic animals which mayhave been genetically engineered to prevent immune-mediated xenograftrejection.

In embodiments where the matrix portion of the cartilage matrix orcartilage growth-enhancing material comprises tissue culture media,without fibrinogen or thrombin, then the biocompatiblechondro-conductive/inductive matrix preferably comprises fibrinogen andthrombin.

In embodiments where the matrix portion of the cartilage matrix orcartilage growth-enhancing material comprises media and fibrinogen, thenthe biocompatible chondro-conductive/inductive matrix preferablycomprises thrombin.

In embodiments where the matrix portion of the cartilage matrix orcartilage growth-enhancing material comprises media and thrombin, thenthe biocompatible chondro-conductive/inductive matrix preferablycomprises fibrinogen.

In different embodiments various combinations of the cartilage matrix orcartilage growth-enhancing material and the biocompatiblechondro-conductive/inductive matrix are possible. By way of non-limitingexample, an embodied composition may comprise juvenile cartilage andthrombin in the cartilage matrix with the biocompatiblechondro-conductive/inductive matrix comprising media and fibrinogen.

In another embodiment the cartilage matrix or cartilage growth-enhancingmaterial may comprise neocartilage and thrombin with the biocompatiblechondro-conductive/inductive matrix comprising media and fibrinogen.

In another embodiment the cartilage matrix or cartilage growth-enhancingmaterial may comprise a combination of juvenile and neocartilage inthrombin with the biocompatible chondro-conductive/inductive matrixcomprising media and fibrinogen.

In any embodiment the compositions may further comprise anosteo-conductive matrix. The osteo-conductive matrix comprises boneparticles. The bone particles may be from any suitable source. Theosteo-conductive matrix may include but not be limited tofibrinogen/thrombin (Tisseel, Crosseal), fibrin/tri-calcium phosphate,fibrin/collagen/tri-calcium phosphate, fibrin/hyaluronate/tri-calciumphosphate PEG base sealants (CoSeal), PEG/tri-calcium phosphate,PEG/collagen (FibroGen) and any of the above components mixed withdemineralized bone matrix. The osteo-conductive matrix may be purchasedfrom commercial sources, such as the demineralized bone matrixcompositions Grafton® (Osteotech, Eatontown, N.J.). Examples of othersources suitable for the osteo-conductive matrix include those disclosedin U.S. Pat. No. 5,356,629, U.S. Pat. No. 6,437,018 and U.S. Pat. No.6,327,257. Suitable compositions may comprise demineralized bone,demineralized bone matrix, nondecalcified bone, cancellous bone orcombinations of the same and a gel material. The osteo-conductive matrixmay also comprise a porous solid, semisolid, paste or gel materialincluding materials such as gelatin, hyaluronic acid, collagen,amylopectin, demineralized bone matrix, and/or calcium carbonatefibrinogen/thrombin, fibrin/tri-calcium phosphate,fibrin/collagen/tri-calcium phosphate, fibrin/hyaluronate/tri-calciumphosphate, in-situ forming PEG hydrogel sealants in-situ forming PEGhydrogel sealants, PEG/tri-calcium phosphate, PEG/collagen,demineralized bone matrix, and any combination thereof.

Osteoconductive materials are generally porous materials and are able toprovide latticework structures such as the structure of cancellous boneor similar to cancellous bone. Such materials may generally facilitateblood-vessel incursion and new bone formation into a defined passivetrellis-like support structure, as well as potentially supporting theattachment of new osteoblasts and osteoprogenitor cells. Osteoconductivematerials may provide an interconnected structure through which newcells can migrate and new vessels can form.

Examples of materials suitable for the osteoconductive matrix includethose disclosed in U.S. Pat. No. 5,356,629 which discloses a compositionof polymethylacrylate biocompatible particles dispersed in a matrix ofcellulose ether, collagen or hyaluronic acid and U.S. Pat. No. 6,437,018which includes a composition of demineralized bone matrix (DBM) in anaqueous carrier that is sodium hyaluronate in a phosphate bufferedaqueous solution. U.S. Pat. No. 6,327,257 discloses compositions withdemineralized bone, nondecalcified bone, cancellous bone and a gelmaterial. There are also compositions that are available commercially,including demineralized bone matrix compositions such as Grafton®(Osteotech, Eatontown, N.J.). These compositions typically comprise aporous solid, semisolid, paste or gel material including materials suchas gelatin, hyaluronic acid, collagen, amylopectin, demineralized bonematrix, and/or calcium carbonate, to create an osteoconductiveenvironment.

In some embodiments the composition optionally further comprises othercomponents or compounds to address the needs of a particular articularcartilage injury or circumstance or a specific patient's individualneeds. By way of non-limiting example the biocompatiblechondro-conductive/inductive matrix may in some instances comprisealbumin, in-situ forming PEG hydrogel, fibrin/hyaluronate,fibrin/collagen/hyaluronate, PEG/hyaluronate, PEG/collagen, other plasmaand protein-based adhesives and sealants, other natural adhesives andsealants and any combination of these.

In any embodiment the cartilage matrix may be distributed throughoutsubstantially all of the biocompatible chondro-conductive/inductivematrix, as shown in FIG. 1. Alternatively the cartilage matrix may bedistributed throughout a portion of the biocompatiblechondro-conductive/inductive matrix, as shown in FIG. 2. The cartilagematrix may be distributed throughout 90% or less of the biocompatiblechondro-conductive/inductive matrix. The cartilage matrix may also bedistributed throughout 80% or less of the biocompatiblechondro-conductive/inductive matrix. The cartilage matrix may also bedistributed throughout 70% or less of the biocompatiblechondro-conductive/inductive matrix. The cartilage matrix may also bedistributed throughout 60% or less of the biocompatiblechondro-conductive/inductive matrix. The cartilage matrix may also bedistributed throughout 50% or less of the biocompatiblechondro-conductive/inductive matrix. The cartilage matrix may also bedistributed throughout 40% or less of the biocompatiblechondro-conductive/inductive matrix. The cartilage matrix may also bedistributed throughout 30% or less of the biocompatiblechondro-conductive/inductive matrix. The cartilage matrix may also bedistributed throughout 20% or less of the biocompatiblechondro-conductive/inductive matrix. The cartilage matrix may also bedistributed throughout 10% or less of the biocompatiblechondro-conductive/inductive matrix.

Similarly, in embodiments where the compositions and methods furthercomprise an osteo-conductive matrix, the osteo-conductive matrix may bedistributed throughout substantially all of the composition.Alternatively the osteo-conductive matrix may be distributed throughouta portion of the composition. It may be desirable in some embodiments tohave the osteo-conductive matrix disposed to contact bone in a defectthat has involvement of both bone and articular cartilage, as shown inFIG. 3. The osteo-conductive matrix may be distributed throughout 90% orless of the composition. The osteo-conductive matrix may also bedistributed throughout 80% or less of the composition. Theosteo-conductive matrix may also be distributed throughout 70% or lessof the composition. The osteo-conductive matrix may also be distributedthroughout 60% or less of the composition. The osteo-conductive matrixmay also be distributed throughout 50% or less of the composition. Theosteo-conductive matrix may also be distributed throughout 40% or lessof the composition. The osteo-conductive matrix may also be distributedthroughout 30% or less of the composition. The osteo-conductive matrixmay also be distributed throughout 20% or less of the composition. Theosteo-conductive matrix may also be distributed throughout 10% or lessof the composition.

In one embodiment a method of use comprises disposing a cartilage matrixof neocartilage or juvenile cartilage, or a combination thereof and abiocompatible chondro-conductive/inductive matrix in any location whererepair or replacement of articular cartilage is desired.

In one embodiment a method of use comprises disposing a cartilage matrixof neocartilage or juvenile cartilage, or a combination thereof and abiocompatible chondro-conductive/inductive matrix and an osteoconductive matrix in any location where repair or replacement ofarticular cartilage is desired. Compositions and methods of theinvention comprising the osteo conductive matrix are useful for repairof replacement of articular cartilage at a site that also includes abone defect.

In other embodiments a method of use comprises disposing any embodimentof the compositions of the invention into a defect and overlaying thecomposition with a retainer. The retainer may be of any suitable sizeand material that functions to maintain the particle in the site wherethe particle(s) is disposed. The retainer may be for example a flap,plug, disc, sheet or patch. In one embodiment the retainer comprises aflap. The flap is made up of either live cells, such as periosteumcells, other natural tissue membrane or synthetic membrane. Theperiosteal flap may be vital or devitalized and may be an autologous oran allograft.

Any of the embodiments of the inventive compositions may be used in anyof the embodiments of the methods of the invention. The compositions maybe extruded or otherwise disposed into the targeted site or configuredinto a device for transplanting into a desired site (FIG. 1). Typicallya multi-unit dispensing device such as a double or triple syringe,joined by a Y-connector, or similar converging connector from thedispensing unit may be used where the components mix as they emerge froma blunt cannula or catheter or other similar device. Any embodiment ofthe compositions may be delivered to the defect site through anarthroscopic portal from a mixing mechanism that automatically metersthe components in the correct ratio, into the desired site for articularcartilage repair or replacement.

Delivery of the compositions may be in a variety of forms andcombinations; by way of non-limiting example the cartilage matrix may bein media and mixed with biocompatible chondro-conductive/inductivematrix comprising fibrinogen and thrombin just prior to use as a 3 partmixture. Alternatively, the cartilage matrix may include thrombin and becombined with a fibrinogen biocompatible chondro-conductive/inductivematrix at the time of use, as a 2 part mixture. In another alternative,the cartilage matrix may include fibrinogen and be combined with abiocompatible chondro-conductive/inductive matrix comprising thrombin atthe time of use, as a 2 part mixture.) By changing the particlesincluded in the matrix, for example the juvenile cartilage pieces, invitro-grown neocartilage and the components that comprise thechondro-inductive matrix and/or the osteo-conductive matrix, the natureof the repair graft can be varied from partial thickness through fullthickness into osteochondral defects, as desired and/or in response theto the specific site where repair or replacement is desired. Anotheralternative for delivery is that various combinations of the cartilagematrix and the biocompatible chondro-conductive/inductive matrix may bepreformed and implanted as a single construct. By way of non-limitingexample, an embodied composition may comprise juvenile cartilagepre-cast in a biocompatible chondro-conductive/inductive matrixcomprising fibrin.

The juvenile neocartilage replacement tissue or pre-cast construct madeup of the juvenile cartilage, a chondro conductive/inductive matrixand/or an osteoconductive matrix can also be attached to naturalcartilage or underlying bone in vivo by sutures or sutureless attachmentsuch as chemical tissue welding or gluing, or by physical attachmentdevices such as tacks or staples. The neocartilage may be grown tovarious size specifications to facilitate implantation.

Any of the compositions may be configured to form a device of thepresent invention and the device may then be implanted, inserted orotherwise suitably disposed in a site where repair or replacement ofarticular cartilage is desired. For example any embodiment of thecompositions may be extruded or delivered into a form or mold to producea specific shape or configuration of device and the produced device maythen be appropriately implanted or otherwise disposed in the site wherereplacement or repair of articular cartilage is desired.

In all cases, the compositions and devices of the invention will have aperiod of plasticity during which they can be implanted and/or molded tothe defect being repaired. These methods of delivery advantageously makeimplantation of the repair articular cartilage possible in a singlearthroscopic procedure, if desired. Once implanted, the cartilagefragments coalesce and replace the matrix with hyaline cartilage tissue.This method can also be extended to neocartilage grown in vitro with theadvantage that some expansion of chondrocytes/neocartilage can be done,generating more repair tissue from a single donation of juvenilecartilage. Juvenile chondrocytes and/or juvenile cartilage/neocartilagecan be combined with the biocompatible matrix using a uniformdistribution as illustrated in FIG. 1 or a non-uniform distribution toincrease the cartilage/chondrocyte density as illustrated in FIG. 2 andFIG. 8, where the cartilage is at a higher density near the bottom ofthe defect.

Similarly, different components can be mixed with the biocompatiblematrix to fill chondral and osteochondral defects. FIG. 3 illustrates apotential usage wherein the bone defect is filled with anosteo-conductive matrix up to the tide mark, above which the chondraldefect is filled with juvenile chondrocytes and/or juvenilecartilage/neocartilage matrix combined with the biocompatible matrix.

The |cartilage| may be harvested from cartilage donors such as juvenileanimals. For example, the donors may be prepubescent humans aged betweenabout 20 weeks and about 13 years. The cartilage may be harvested from avariety of cartilage sites, including facing surfaces of bonespositioned at articulating joints. Among particularly desirable harvestsites are |femoral| condyles, tibial plateaus and interior surfaces ofpatella. To harvest the cartilage, the harvest sites are exposed. Thesurface of a harvest site is scored with a blade such as a #10 scalpelhaving a ceramic coated edge (e.g., an IonFusion scalpel blade availablefrom IonFusion Surgical, a division of Molecular Metallurgy, Inc. of ElCajon, Calif.) Although the site may be scored in other patterns withoutdeparting from the scope of the present invention, in one embodiment thesite is scored in a square grid pattern having sides measuring about onemillimeter. Further, although the site may be scored to other depthswithout departing from the scope of the present invention, in oneembodiment the site is scored to a depth of between about one millimeterand about three millimeters or more. Once the site is scored, at least aportion of the scored cartilage is separated from underlying bone, suchas by shaving the scored surface with the aforementioned scalpel. Aswill be appreciated by those skilled in the art, separating thecartilage in this fashion results in small generally cube-shapedparticles of cartilage having sides of about one millimeter. Tissueother than cartilage, such as vascularized bone and tendons, generallyshould be avoided when separating the cartilage from the bone.

The separated particles are collected in a container such as a conicaltube. The particles may be stored in or rinsed with a saline solutionsuch as a 0.9% saline solution. After rinsing or storage, the salinesolution may be removed from the particles by aspiration and anotherpreservative may be added to the particles. For example, a storagesolution comprising hydroxyethyl starch (50 g/L)m lactobionic acid (35.8g/L), adenosine (1.34 gL), NaOH (5M) (5 mL/L), KH2PO4 (3.4 g/L), MgSO4(0.6 g/L), glutathione (0.92 g/L), raffinose (17.8 g/L), and KOH (5M)(pH to 7.4) may be added to the particles.

A kit for repairing cartilage may be formed using the particles.Generally, the kit includes an outer bag or pouch having a hollowinterior, a sterile container positioned in the hollow interior, andcartilage particles positioned in a receptacle of the container.Although the outer pouch may have other configurations without departingfrom the scope of the present invention, in one embodiment the pouch isformed from two sheets, each of which has a central portion surroundedby a margin. The sheets are separably joined to one another at theirmargins. One such pouch is available from Amcor Flexibles Healthcare ofAnn Arbor, Mich., and is identified as an RLP-041 HS pouch made from a48 ga PET/10 lb LDPE/2 mil peelable film (LFM-101). The pouch is about4×6 inches and has 15 degree chevron configuration with thumb notch. Inone embodiment, the container includes a tray having a teardrop-shapedcentral cup or receptacle and a lip or flange surrounding thereceptacle. One such container is available from Prent Corporation ofJanesville, Wis., and is formed from a laminate comprising a Glidexsheet sandwiched between PETG sheets having an overall thickness ofabout 0.020 inch. A removable cover is attached to the lip of the trayfor sealing the receptacle to retain the particles in the receptacle.One such cover is available from Tolas Health Care Packaging ofFeasterville, Pa., and is known as a TPC-0777A peelable lamination for|sterile∥device|packaging. Although the cover may have other dimensionswithout departing from the scope of the present invention, in oneembodiment the cover has a thickness of about 3.95 mils and is about1.57×3.15 inches.

In one embodiment, excess liquid is removed from the particles byaspiration and a 50 mg scoop is used to measure a desired quantity ofparticles into a sterile tray, a desired measure of preservativesolution (e.g., 2.5 mL) is added to the tray and the cover is sealedagainst the rim of the tray to close the container. The container isloaded into a pouch and the pouch is sealed for storage and transport.Once ready for use, the pouch is pealed open and the container isdeposited in a sterile environment. The non-sterile pouch is disposedand the container is opened by peeling back the cover to expose theparticles of cartilage.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following specific examples are offered by wayof illustration only and not by way of limiting the remainingdisclosure.

Example 1 Juvenile Human Articular Cartilage (JHAC) in a HyaluronateMatrix

In certain embodiments of the composition as described hereinparticulate JHAC was embedded within a hyaluronate hydrogel andevaluated for their viscosity and their ability to adhere within adefect. Hyaluronate forms a viscous gel that can hold the cartilageparticles within a defect during implantation. Concentrations ofhyaluronate ranging from 5 mg/ml to 100 mg/ml were tested in thisexample. In the mixture illustrated by FIG. 4, JHAC was embedded in agel containing 50 mg of hyaluronate dissolved in 1 ml of phosphatebuffered saline. Although suitable for a matrix, hyaluronate alonelacked cross-linking within the gel. Therefore, in one preferredcomposition, a component such as fibrin is included to retard or preventdissolution of the chondro-conductive matrix.

Example 2 In Vitro JHAC Re-Integration

When juvenile tissue is maintained in the laboratory embedded within afibrin matrix, the tissue has the ability to re-integrate. In thisexperiment, JHAC was minced and cast in human fibrinogen within acylindrical mold and then cultured for 60 days in a standard cellculture using a proprietary serum-free medium, developed at IstoTechnologies, Inc. The tissue composite was then fixed and histologicalslides were prepared and stained with Safranin-O which stains red in thepresence of sulfated glycosaminoglycan (S-GAG). Safranin-O staining isunique to the hyaline cartilage that lines the articular surfaces of thejoints. As shown in FIG. 5, the two pieces of tissue have begun tointegrate with each other in the fibrin-filled space between theoriginal tissue pieces. The dark red stain (original proof) indicatesthat the tissue has remained viable and is maintaining a normalhyaline-cartilage phenotype with regard to S-GAG composition.

Example 3 Minced JHAC Implantation

Minced JHAC was implanted into Spanish goats using the methods of theinvention, further demonstrating the usefulness of the invention. A six(6) mm circular defect was created in the weight-bearing region of theright, medial femoral condyle. Minced juvenile human articular cartilagewas placed into the defect which was subsequently filled with humanfibrin and covered with a live periosteal flap sutured into thesurrounding cartilage. The limb was then set in a modified Thomas splintfor a period of six weeks during which the animal was able to ambulatewithout exposing the repaired site to full weight-bearing forces.

FIG. 6 shows the repaired medial femoral condyle (right side ofphotograph) six weeks after implantation. The surface of the repair siteappears relatively smooth and the tissue has been retained within theoriginal defect. A 1 mm thick section through the defect site is shownin FIG. 7. The section shows that the defect is filled with a white,translucent material including the original tissue pieces. Fluorescentprobes stain the nuclear DNA red and identify dead cells while greenprobes stain living cells within the cartilage matrix. The juvenilecartilage is embedded into a chondro-conductive matrix composed offibrin that is less cellular. Microscopic examination of the sectionusing a viability-indicating stain indicates that both the originaltissue and cells that have migrated into the fibrin matrix stain green(original proof) and are therefore viable (FIG. 8).

Safranin-O stained histological sections indicate that the defect siteis populated not only by the original implanted tissue, but also bycells that have migrated into the defect site as illustrated by FIG. 9.The original tissue retains the red stain (original proof) indicatingS-GAG in the extracellular matrix while the cell-populated matrixsurrounding the transplanted tissue has not yet been replaced with ahyaline-like extracellular matrix. The juvenile cartilage is embeddedinto a chondro-conductive matrix composed of fibrin.

These data demonstrate the successful repair of a chondral defect with aviable tissue construct containing juvenile hyaline cartilage accordingto one embodiment of the present invention.

Other Embodiments

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Particular formulations and processes of the presentinvention are not limited to the descriptions of the specificembodiments presented, but rather the descriptions and examples shouldbe viewed in terms of the claims that follow and their equivalents.While some of the examples and descriptions above include someconclusions about the way the invention may function, the inventors donot intend to be bound by those conclusions and functions, but puts themforth only as possible explanations.

It is to be further understood that the specific embodiments of thepresent invention as set forth are not intended as being exhaustive orlimiting of the invention, and that many alternatives, modifications,and variations will be apparent to those of ordinary skill in the art inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and scope ofthe following claims.

REFERENCES CITED

All publications, patents, patent applications and other referencescited in this application are herein incorporated by reference in theirentirety as if each individual publication, patent, patent applicationor other reference were specifically and individually indicated to beincorporated by |reference|.

What is claimed is:
 1. A composition comprising minced juvenilecartilage particles and a retainer.
 2. The composition of claim 1,further comprising a matrix.
 3. The composition of claim 2, wherein thematrix comprises a biocompatible chondro-inductive matrix.
 4. Thecomposition of claim 2, wherein the matrix comprises a biocompatiblechondro-conductive matrix.
 5. The composition of claim 1, wherein thejuvenile cartilage particles comprise articular cartilage.
 6. Thecomposition of claim 1, wherein the juvenile cartilage particles includeone dimension of 1 to 3 mm.
 7. The composition of claim 1, wherein thejuvenile cartilage particles are 1 to 27 mm³.
 8. The composition ofclaim 1, wherein the juvenile cartilage particles are from a human donorless than fifteen years of age at the time of donation.
 9. Thecomposition of claim 1, wherein the juvenile cartilage particles arefrom a human donor less than two years of age at the time of donation.10. The composition of claim 1, wherein the juvenile cartilage particlesare from a human donor that is prepubescent at the time of donation. 11.The composition of claim 1, wherein the juvenile cartilage particleshave a surface-to-volume ratio greater than
 1. 12. The composition ofclaim 1, wherein the juvenile cartilage particles are from a donornon-autologous to a recipient.
 13. The composition of claim 2, whereinthe juvenile cartilage particles are distributed within about 60% orless of the matrix.
 14. The composition of claim 2 wherein the juvenilecartilage particles are distributed within about 20% or less of thematrix.
 15. The composition of claim 1, wherein the retainer is selectedfrom the group consisting of a flap, a plug, a disc, a sheet and apatch.
 16. The composition of claim 1, wherein the retainer comprises aperiosteal flap.
 17. The composition of claim 1, wherein the retainercomprises a synthetic membrane.
 18. The composition of claim 1, whereinthe retainer comprises a sutureless attachment.
 19. The composition ofclaim 2, wherein the matrix comprises fibrin.
 20. The composition ofclaim 2, wherein the matrix comprises polyethylene glycol.
 21. Thecomposition of claim 2, wherein the matrix is selected from the groupconsisting of plasma-based adhesives or sealants, protein-basedadhesives or sealants, natural adhesives or sealants, and anycombination thereof.
 22. The composition of claim 2, wherein the matrixcomprises an osteo-conductive matrix.
 23. The composition of claim 2,wherein the matrix comprises a media suitable for tissue or cellculture.